Project Summary Glia have emerged as important contributors to spinal motor neuron survival; however, the mechanisms by which they promote motor neuron viability are not well understood. This proposal will examine a novel pathway in oligodendrocytes that regulates the survival of spinal motor neurons in the adult nervous system. This pathway includes the six-transmembrane enzyme GDE2 (glycerophosphodiester phosphodiesterase 2; GDPD5), which cleaves the GPI (glycosylphosphatidylinositol)-anchor that links proteins to the cell-membrane, and the GPI- linked heparan sulfate proteoglycan Glypican (GPC) 6. In support of this pathway, Gde2 knockout (KO) mice show progressive motor neuron degeneration that culminates in motor neuron loss and motor deficits. Timed and cell-specific genetic ablation of GDE2 associates motor neuron pathologies with postnatal GDE2 function and additionally, pinpoints GDE2 cellular requirement for motor neuron survival to oligodendrocytes. Unbiased screens to isolate GPI-anchored GDE2 substrates combined with biochemical evaluation in an independent model of motor neuron degeneration, identify GPC6 as a potential mediator of GDE2 function in motor neuron survival. GDE2 releases GPC6 through GPI-anchor cleavage, and cell-based assays identify a second GDE2- dependent mechanism of GPC6 release by extracellular vesicles (EVs). Further, conditioned medium containing cleaved and EV GPC6 rescues motor neuron loss of Gde2 KO organotypic spinal cord cultures. These observations suggest the hypothesis that GDE2 constitutes a physiological pathway in oligodendrocytes that promotes spinal motor neuron survival, and that GDE2 neuroprotective function is mediated through release and delivery of GPC6. Proposed experiments will test this hypothesis in two specific aims. Aim 1 will define properties and specificity of EV and cleaved GPC6 to motor neuron survival in context of the GDE2 pathway, and explore the contribution of released GPC6 to motor neuron survival in physiological and pathological settings. Aim 2 will determine consequences of GDE2 loss in oligodendrocytes on GPC6 release, and will examine physiological requirement of GPC6 expression in oligodendrocytes to motor neuron survival. Outcomes from the proposed studies are expected to identify the GDE2-GPC6 pathway as essential for motor neuron viability, and to provide new mechanistic insight into oligodendrocyte contributions to neuronal survival.